Rotavirus is the major cause of severe, life-threatening gastroenteritis in young children and animals. Rotaviruses are large (1000 A), complex, icosahedral assemblies. This virus has been the subject of extensive biochemical, genetic and structural studies because of its medical relevance, intriguing structural complexity, and unique strategies of replication and morphogenesis. Rotaviruses contain 11 segments of double-stranded RNA encapsidated within three concentric capsid layers. Of the 12 proteins encoded by the genome, six are structural (VP1-7), and six are non-structural (NSP1-6). In the last three and half years, we have made exciting new discoveries that provide a better characterization of rotavirus structure and a deeper insight into the structural basis of various virus functions such as trypsin-enhanced infectivity, virus assembly, endogenous transcription, and genome replication and packaging. These recent developments, have allowed us to plan more in-depth dissection of structure-function correlations in rotavirus using a combination of highresolution cryo-EM, X-ray crystallographic, and biochemical techniques. The specific objectives of this ongoing project are: 1) To further investigate the mechanism of protease-enhanced infectivity and spike assembly, and conformational changes associated with cell entry of rotavirus using in vitro recombinant VLPbased techniques and electron cryo-tomographic approaches. 2) To further our understanding of the structural basis of endogenous transcription in rotavirus by characterizing the structural alterations in response to transcriptional activation using high-resolution cryo-EM techniques. 3) To dissect the structural mechanisms of regulation of rotavirus genome replication/packaging using X-ray crystallographic and single particle cryo-EM analysis of NSP2 with its various ligands such as RNA, NSP5, and VP1. 4) To explore further the role of the recently discovered novel NDP kinase activity in genome replication and to carry out structure-based design of inhibitors of this activity. 5) To construct an in vitro replication complex based on our hypothesis that capsid assembly and genome encapsidation in rotavirus are concomitant processes initiated by pentamers of dimers of VP2. 6) To carry out structural studies on rotavirus NSP1, a rotavirus non-structural protein that interferes with the interferon response pathway. Our structural information, in conjunction with continued advances in molecular virology of rotavirus, have the potential to enhance the development of more effective methods of disease prevention and control. More importantly, we expect to continue to discover new fundamental structural information to help understand how these complex viruses gain entry into host cells, assemble, transcribe, replicate, and package their genomes.